Over the past decade, our group has been actively engaged in development of engineered cell lines as potential alternatives to insulin injection or islet transplantation for insulin replacement in insulin dependent diabetes mellitus (IDDM). A major hurdle faced in bringing such an approach to fruition is to provide protection against immune destruction of the transplanted cell lines. This application seeks to develop a combinatorial approach for overcoming this obstacle. The first part of our strategy is to use genetic engineering approaches to provide protection against the cytotoxic effects of inflammatory cytokines such as IL-1beta and gamma-IFN that are known to be involved in beta-cell destruction in IDDM. We have recently shown that overexpression of MnSOD in INS-1 insulinoma cells provides complete protection from IL-1beta-mediated cytotoxicity and also blocks iNOS upregulation by the cytokine. The first specific aim seeks to understand the mechanism of this protective effect of MnSOD by a combination of biochemical and molecular approaches. MnSOD overexpressing cells are not protected from the cytotoxic effects of gamma-IFN. Thus, the second specific aim seeks to define and discriminate the pathways by which gamma-IFN and IL-1beta exert their cytotoxic effects. We will also engineer insulinoma cell lines for expression of the anti-apoptotic gene bcl-2, and test whether this maneuver provides protection against one or both of the cytokines. The third and final specific aim will involve transplantation of the new cell lines engineered for resistance to cytokine damage into rodents. These experiments will include the second part of our strategy, which is to encapsulate the engineered cell lines in a cell-impermeable device provided by industrial collaborators that will prevent contact between cellular elements of the immune system and the transplanted cells. The broad goal of the application is to determine whether this combinatorial approach will allow long-term survival of transplanted, insulin producing cells in rodent allograft and IDDM models. If we are successful, these studies could lead to development of viable strategies for cell-based hormone replacement in IDDM and other diseases.